Alopecia Areata (AA) is one of the most common autoimmune diseases in the US, with a lifetime risk of 1.7%, it affects approximately 5.3 million individuals across all ethnic groups. We recently carried out a genome-wide association study (GWAS) to identify common alleles that contribute to risk of AA, and identified several genomic regions harboring potential susceptibility genes. In our Preliminary Studies, we have generated a significant and unique resource of genomic and genetic datasets in alopecia areata patient samples using several approaches, including GWAS genotyping on >1000 individuals, ImmunoChip on >400 individuals, Linkage analysis on >50 families, Gene expression profiling on >100 individuals, Targeted resequencing in GWAS regions in 124 individuals, and Whole exome sequencing in >10 alopecia areata probands selected from linkage families. These robust and highly integrated datasets provide a rich foundation from which to interrogate the functional significance of variants in AA candidate genes in the context of this innovative proposal. As a first step towards understanding the biological significance of these results, we must now perform deep sequencing analysis in these regions to identify causal variants that are driving the association of tagSNPs. The emerging picture of the genetic architecture of common diseases contains niches for both common and rare variants acting independently or in concert to influence phenotypes. Understanding the impact of these variants in disease pathogenesis is the first key step in moving toward novel therapies for AA. These studies are extremely timely, and will allow us to place AA into the context of other autoimmune diseases in which GWAS and deep sequencing is already underway. We postulate that causal variants in candidate genes underlie the susceptibility to develop AA. This grant is focused on carrying out deep sequencing studies to identify new variants in candidate genes, followed by functional genomics of variants in several in vitro contexts, which will allow us to determine: 1) the nature of the specific variats contributing to AA susceptibility; and 2) the mechanism(s) by which they contribute to disease pathogenesis. In this proposal, we will carry out Exome-plus sequencing with enrichment in our previous GWAS regions to identify causal variants. We will then interrogate the functional consequences of variants within candidate genes expressed in both the hair follicle and immune cells, with an emphasis on the NKG2D pathway.